Chemically hypoxic conditioned mesenchymal extracellular vesicles for treating viral lung injury

Abstract

This invention provides a novel cell-free therapy using extracellular vesicles (EVs) derived from chemically hypoxia-preconditioned umbilical cord mesenchymal stromal cells (UC-MSCs). In physiologically relevant in vitro lung injury models, hypoxic MSC-EVs significantly restored acute lung capacity (AFC), reduced active inflammatory response (APP), inhibited pro-inflammatory cytokines, and enhanced ion transporter activity more effectively than normoxic EVs. In vivo, hypoxic MSC-EVs also improved survival rates and reduced viral load in mice infected with highly pathogenic influenza A (H5N1). This invention demonstrates that hypoxia preconditioning significantly enhances the therapeutic efficacy of MSC-EVs against viral lung injury, thus providing an innovative and safer alternative to live-cell therapy, particularly beneficial for treating advanced acute lung injury in immunocompromised patients, and representing an advancement over previous MSC- or EV-based strategies.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 730,919, filed December 11, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the fields of regenerative medicine and the treatment of respiratory diseases, and more specifically to a method for treating acute lung injury using extracellular vesicles derived from hypoxia-preconditioned mesenchymal matrix cells. Background Technology

[0003] Acute lung injury (ALI), particularly in cases caused by severe viral infections such as highly pathogenic influenza viruses (e.g., H5N1), represents a serious clinical condition characterized by impaired alveolar fluid clearance (AFC), increased alveolar protein permeability (APP), excessive inflammation, and impaired gas exchange. These pathological changes typically lead to respiratory failure and are associated with high morbidity and mortality. Current treatment for viral ALI primarily focuses on supportive care, including antiviral drugs, mechanical ventilation, and corticosteroids. However, the efficacy of these treatments is limited, especially in the later stages of disease progression when irreversible lung damage has occurred. Furthermore, existing therapies mainly focus on suppressing viral replication or alleviating symptoms but fail to effectively promote tissue repair or modulate the host's inflammatory response.

[0004] Mesenchymal stromal cells (MSCs) have emerged as promising candidate cell types in regenerative medicine due to their immunomodulatory effects, low immunogenicity, and ability to promote tissue repair. In recent years, research focus has shifted from directly utilizing MSCs themselves to leveraging their paracrine signaling components, collectively known as the MSC secretome, which includes soluble cytokines and extracellular vesicles (EVs). These EVs have been shown to mediate key therapeutic effects by transferring bioactive molecules to recipient cells. Despite these advantages, unmodified MSCs or EVs derived from normoxic cultures may exhibit inconsistent therapeutic outcomes due to their variable composition and limited potency.

[0005] It has been reported that pre-modulated MSCs under hypoxic conditions can enhance the therapeutic efficacy of the resulting secretosomes, as these hypoxic conditions mimic the ischemic microenvironment commonly found in damaged tissues. To induce hypoxic conditions, in addition to physically reducing the oxygen concentration in the culture environment using a hypoxic chamber, hypoxia mimics can be used to prevent the degradation of hypoxia-inducible factor (HIF), providing greater oxygen stress stability. Specifically, hypoxia-induced EVs are rich in angiogenesis and regeneration factors, which can potentially enhance tissue repair and immune regulation. However, current research on hypoxia-modulated MSC-EVs for treating acute lung injury caused by respiratory viruses remains insufficient, particularly lacking studies on physiologically relevant models that accurately reflect the pathophysiology of human diseases. Furthermore, the mechanisms underlying the enhanced therapeutic effects of such EVs are not yet fully characterized.

[0006] Therefore, there is a need in the art for a safe, effective, and cell-free therapeutic strategy for treating acute lung injury caused by viral infections. Such a strategy should address the limitations of conventional MSC-based therapies, including the risk of immune rejection, tumorigenicity, and manufacturing complexity. Ideally, the approach would reduce inflammation, support epithelial repair, and enhance antiviral responses, while avoiding the safety and regulatory challenges of live cell transplantation. Summary of the Invention

[0007] Despite the recognized therapeutic potential of mesenchymal stromal cells (MSCs), their clinical application remains hampered by several limitations, including the risk of immune rejection, the tumorigenic potential of undifferentiated cells, and the possibility of metastasizing contaminated or infected cell cultures. Furthermore, in vitro expansion of MSCs can lead to phenotypic drift and loss of functional potency. These safety and stability concerns limit the direct use of MSCs in cell-based therapies for respiratory infectious diseases such as influenza-induced acute lung injury.

[0008] In view of these drawbacks, the present invention aims to provide a safer and more effective treatment strategy that utilizes the regenerative and immunomodulatory properties of MSCs without requiring live cell transplantation. Specifically, the present invention utilizes EVs secreted by MSCs, which retain many of the therapeutic functions of the parent cells while avoiding complications associated with cell therapy. The present invention addresses the above-mentioned need by providing a novel use of chemically hypoxia-preregulated MSC-derived EVs and validating its efficacy using both in vitro and in vivo models of influenza-induced lung injury.

[0009] This invention discloses a method for enhancing the therapeutic efficacy of MSC-derived EVs by pre-regulating umbilical cord-derived MSCs (UC-MSCs) under hypoxic conditions prior to EV isolation. Cobalt(II) chloride (the hypoxia mimic used herein) prevents the rapid degradation of HIF-1α after removal of the cobalt-containing medium. This allows for extended experimental manipulation of hypoxic cells by several hours, overcoming the problem of rapid HIF-1α degradation caused by reoxygenation upon opening the hypoxic chamber. These hypoxia-pre-regulated EVs (hypoxic MSC-EVs) exhibited excellent regenerative activity in both in vitro and in vivo models of respiratory virus-induced lung injury. A physiologically relevant human lung injury model was established using highly pathogenic influenza A (H5N1) virus to elucidate the protective mechanisms of EVs against alveolar fluid clearance, alveolar protein permeability, inflammatory cytokine inhibition, ion transporter restoration, and viral replication inhibition.

[0010] In a first aspect, the present invention provides an EV composition comprising a population of EVs isolated from one or more mesenchymal stromal cells (MSCs) cultured under chemically induced hypoxic conditions. Prior to collection, the one or more MSCs are exposed to a hypoxia mimicry agent, the EVs exhibit a particle size distribution in the range of approximately 30 nm to approximately 150 nm in diameter, and the EVs are characterized by a lipid bilayer membrane encapsulating cytoplasmic protein cargo.

[0011] According to one embodiment of the present invention, the cytoplasmic protein cargo includes at least CD9 and HIF-1α, and does not contain GM130.

[0012] According to one embodiment of the present invention, the MSC is an umbilical cord-derived mesenchymal stromal cell.

[0013] According to one embodiment of the invention, the one or more MSCs are exposed to 50 μM to 200 μM cobalt(II) chloride for 48 to 96 hours. Alternative hypoxia mimics, such as deferoxamine (50 μM to 100 μM) and dimethyloxaloylglycine (0.5 mM to 1 mM), may also be used.

[0014] According to one embodiment of the present invention, the EV is obtained from the conditioned medium by sequential ultracentrifugation and 0.22 μm membrane filtration.

[0015] According to one embodiment of the present invention, the EV includes at least one protein selected from the group consisting of APOE, FGG and NPTX1, wherein the expression level of at least one protein is at least 2-fold higher than that of EV derived from normoxic cultured MSCs.

[0016] According to one embodiment of the present invention, when the EV acts on alveolar epithelial cells infected with H5N1, the mRNA expression level of at least one cytokine selected from TNF-α, IFN-β and RANTES is reduced by at least 2-fold relative to untreated infected cells.

[0017] According to one embodiment of the present invention, the expression of ion transport proteins selected on the EV from the group consisting of: epithelial sodium channels (ENaC), CFTR, and Na+. + / K + ATPase.

[0018] According to one embodiment of the present invention, the EV induces at least 2-fold ISG15 mRNA expression in virus-infected alveolar epithelial cells.

[0019] According to one embodiment of the invention, the EV is further formulated in a pharmaceutically acceptable carrier suitable for intravenous administration.

[0020] According to one embodiment of the present invention, the EV is formulated into a dosage form selected from the group consisting of: The freeze-dried powder contains one or more cryoprotectants selected from the group consisting of trehalose, sucrose, and mannitol; Atomizable aqueous solution, wherein the atomizable aqueous solution is formulated for lung administration; and One or more polymeric microparticles, the one or more polymeric microparticles comprising the EV encapsulated in a biodegradable polymer selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), chitosan, and hyaluronic acid.

[0021] On the other hand, the present invention provides a method for treating acute lung injury in a subject in need, the method comprising administering an effective amount of an EV composition to the subject, wherein the lung injury is caused by a viral infection, and wherein the EV is derived from MSCs cultured under chemically induced hypoxia using a hypoxia mimic.

[0022] According to one embodiment of the present invention, the MSC is exposed to 50 μM to 200 μM cobalt(II) chloride for 48 to 96 hours.

[0023] According to one embodiment of the present invention, the EV composition is administered via intravenous, intranasal, or intratracheal routes.

[0024] According to one embodiment of the present invention, the EV can reduce the viral load in lung tissue by at least 50% compared to the untreated control group.

[0025] According to one embodiment of the present invention, the EV reduces the expression levels of TNF-α and RANTES by at least 2-fold compared to the untreated control group.

[0026] According to one embodiment of the present invention, the EV restores alveolar fluid clearance (AFC) and reduces alveolar protein permeability (APP) within 24 hours after administration.

[0027] According to one embodiment of the present invention, the EV increases ISG15 expression in infected alveolar epithelial cells by at least 2-fold.

[0028] According to one embodiment of the present invention, the EV composition is administered in combination with one or more antiviral agents, the one or more antiviral agents comprising oseltamivir, zanamivir, or ribavirin.

[0029] According to one embodiment of the present invention, the effective amount of the EV composition comprises approximately 1 × 10⁻⁶ per application. 8 Each EV particle up to 1×10 12 Dosage of each EV particle.

[0030] According to one embodiment of the present invention, the EV composition is administered in multiple doses over a period of 3 to 10 days after the onset of viral infection symptoms.

[0031] Surprisingly, compared with normoxic EVs, hypoxic MSC-EVs showed significantly improved therapeutic efficacy, including a greater reversal of H5N1-induced AFC damage, enhanced recovery of ion transporter activity, stronger upregulation of antiviral gene expression, and more effective suppression of inflammatory cytokines and viral load. These significant effects were further validated in a mouse model infected with H5N1, where hypoxic MSC-EV treatment significantly improved survival outcomes and reduced pathological weight loss.

[0032] Compared to existing methods that directly utilize unmodified EVs or MSCs, this invention demonstrates that premodulation of UC-MSCs under chemically induced hypoxic conditions (e.g., using cobalt(II) chloride) significantly alters EV protein cargo, resulting in significant therapeutic advantages. These advantages include enhanced inhibition of pro-inflammatory cytokines (e.g., TNF-α, IFN-β), improved restoration of ion transporters (e.g., ENaC, CFTR), and stronger upregulation of antiviral genes (e.g., ISG15), effects not anticipated in the prior art. Attached Figure Description

[0033] Embodiments of the invention are described in more detail below with reference to the accompanying drawings, in which:

[0034] Figure 1A-1D The isolation of MSC-EVs from cell culture medium is shown. Figure 1A A schematic diagram illustrating the separation of MSC-EVs from anoxically treated MSC medium containing secreted EVs and proteins using differential centrifugation. Figure 1B Western blotting of proteins, Figure 1C Nanoparticle tracking analysis and ( Figure 1D EVs were characterized by transmission electron microscopy. N = normoxic, H = hypoxic, GM130 = negative EV marker, CD9 = positive EV marker, β-actin = loaded control, and HIF-1α = hypoxia marker.

[0035] Figure 2A-2G This study demonstrated that, in vitro, hypoxia-regulated MSC-EVs were more effective than normal MSC-EVs in preventing damage to alveolar epithelial AFC and APP caused by influenza A (H5N1) virus. Figure 2A This is a schematic diagram of an in vitro lung injury model used to study AFC and APP. AECs seeded in transwells were first infected with H5N1 influenza virus and then treated with normoxic or hypoxic MSC-EVs. Compared to untreated infected cells, EV treatment of infected cells restored AFC (AFC). Figure 2B ) and reduced the APP ( Figure 2C Cells infected with H5N1 showed overexpression of pro-inflammatory cytokines. Figure 2D ) and inhibition of ion transporter activity ( Figure 2E ); EV treatment reverses these effects. Figure 2F Further upregulation of antiviral genes after post-infection EV treatment may be due to EV suppressing infectious viral titers. Figure 2G The reason for this is that the data represent the mean ± SD from the three experiments. *P≤0.05, **P≤0.01 and ***P≤0.001.

[0036] Figure 3A-3G The study showed that hypoxic MSC-EVs significantly reduced weight loss in mice infected with H5N1. Figure 3A A schematic diagram of intranasal H5N1 infection in mice and subsequent treatment with intravenous administration of MSC-EV. Figure 3B Hypoxic MSC-EV significantly prevented H5N1-induced weight loss. Figure 3C Treatment with MSC-EV significantly improved the survival rate of mice infected with H5N1. MSC-EV inhibited ( Figure 3D Lung homogenate and ( Figure 3E The levels of pro-inflammatory cytokines in bronchoalveolar lavage (BAL) fluid. For example ( Figure 3FInfluenza virus M gene expression and ( Figure 3G TCID assays showed that MSC-EV treatment significantly reduced infectious viral titers in mice. Data represent mean ± SD from three experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 and ****P ≤ 0.0001.

[0037] Figures 4A-4C The study demonstrates the properties of hypoxia-regulated MSC-EVs revealed by proteomic analysis compared to normoxic MSC-EVs. Figure 4A Compared to normoxic MSC-EVs, hypoxic MSC-EVs showed upregulated and downregulated proteins. Figure 4B Cellular components associated with hypoxic MSC-EV proteins. Figure 4C (Molecular functions associated with hypoxic MSC-EV proteins) Detailed Implementation

[0038] definition

[0039] Throughout this specification, unless the context otherwise requires, the word “comprise” or variations thereof, such as “comprises” or “comprising”, should be understood to imply inclusion of the stated whole or group of wholes, but not to exclude any other whole or group of wholes. It should also be noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” and “comprising” may have the meaning attributed to them under U.S. patent law, for example, allowing for elements not expressly listed but excluding elements present in the prior art or affecting the essential or novel features of the invention.

[0040] Furthermore, throughout this specification and claims, unless the context otherwise requires, the word "include" or variations thereof, such as "includes" or "including," shall be understood to imply inclusion of the stated whole or group of wholes, but not to exclude any other whole or group of wholes.

[0041] As used herein and unless otherwise defined, the terms “substantially,” “basically,” “approximately,” and “about” are used to describe and explain small variations. When used in conjunction with an event or situation, the terms may cover instances where the event or situation occurred precisely or instances where the event or situation was close to occurring. For example, when used in conjunction with a numerical value, the terms may cover a range of variation less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%.

[0042] References to "an embodiment," "an example embodiment," "exemplary embodiment," etc., in this specification indicate that the described embodiment may include a specific feature, structure, or characteristic; however, not every embodiment necessarily includes that specific feature, structure, or characteristic. Furthermore, such phrases do not necessarily refer to the same embodiment. Moreover, when a specific feature, structure, or characteristic is described in connection with an embodiment, it is assumed that the effect of such feature, structure, or characteristic in conjunction with other embodiments is within the knowledge of those skilled in the art, whether explicitly described or not.

[0043] As used herein, the term "extracellular vesicle" (EV) refers to a nanoscale vesicle naturally secreted by cells into the extracellular space and enclosed by a lipid bilayer. The EVs of this invention typically have a diameter ranging from approximately 30 nanometers to 150 nanometers, exhibit a spherical morphology, and are substantially free of organelle contaminants. EVs may contain proteins, RNA, lipids, or other bioactive molecules derived from the original cell and are capable of modulating the bioactivity of the recipient cell.

[0044] The term “hypoxia-preconditioned MSCs” or “hypoxia-MSCs” refers to mesenchymal stromal cells that have been cultured under chemically induced hypoxia conditions (e.g., by exposure to 50 μM to 200 μM cobalt(II) chloride for 48 to 96 hours, thereby mimicking hypoxia conditions and enhancing the secretion of extracellular vesicles rich in therapeutic factors).

[0045] The term "pharmaceutically acceptable carrier" refers to any biocompatible medium or excipient that can be used to formulate extracellular vesicle compositions for therapeutic administration without causing significant adverse effects. Exemplary carriers include, but are not limited to, saline, phosphate-buffered saline (PBS), aqueous buffers, cryoprotectant solutions, or biodegradable polymers.

[0046] The term "acute lung injury" (ALI) refers to a pathological condition of the lungs characterized by impaired alveolar fluid clearance (AFC), increased alveolar protein permeability (APP), epithelial barrier dysfunction, and an excessive inflammatory response, usually caused by infection with respiratory viruses such as influenza A (H5N1).

[0047] The term "effective amount" refers to the amount of extracellular vesicle composition sufficient to produce a measurable biological response or therapeutic benefit (such as reduced inflammation, improved AFC, suppressed viral load, or increased survival) in the treated subject.

[0048] Other definitions of the selected terms used herein can be found within the specific embodiments of the invention and apply throughout. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0049] This invention provides a novel cell-free treatment method for treating acute lung injury caused by respiratory pathogens. By utilizing hypoxia-induced enhancement of MSC-EV function, this invention overcomes the limitations of conventional MSC-based therapies.

[0050] Respiratory viral infections caused by highly pathogenic strains (such as influenza A (H5N1)) are associated with high mortality rates and limited treatment options. Existing antiviral therapies are often ineffective in preventing disease progression, especially when treatment is initiated in later stages of the disease. Survivors often suffer from long-term pulmonary sequelae due to damage to the epithelial barrier, including symptoms such as decreased lung function and persistent fatigue. Therefore, there is an urgent need for a treatment that promotes the functional recovery of alveolar structure.

[0051] Extracellular vesicles (EVs) are lipid bilayer-enclosed nanovesicles naturally secreted by living cells. These vesicles play a role in intercellular communication by transferring bioactive cargoes such as proteins, RNA, and lipids to recipient cells. The uptake of EV contents alters intracellular signaling and modulates recipient cell phenotypes. Specifically, EVs derived from mesenchymal stromal cells (MSC-EVs) have demonstrated immunomodulatory, anti-inflammatory, and tissue-repairing effects in various preclinical models. Their application as cell-free therapeutic agents is particularly attractive due to their low immunogenicity and stability.

[0052] Therefore, this invention relates to a therapeutic composition comprising EVs derived from mesenchymal stromal cells (MSCs) pre-regulated under hypoxic conditions. The composition is suitable for treating acute lung injury (ALI) caused by respiratory viral infections. Specifically, this invention provides a cell-free therapeutic approach that modulates pro-inflammatory responses, restores epithelial ion transport, and enhances antiviral gene expression in damaged lung tissue.

[0053] In one embodiment, MSCs were subjected to hypoxia preconditioning by chemical induction with 50 μM to 200 μM cobalt(II) for 48 to 96 hours. This hypoxia exposure enhanced the secretory activity of MSCs and altered the composition of the EVs released. EVs obtained from hypoxia-preconditioned MSCs (hypoxia-MSC-EVs) exhibited different proteomic characteristics and enhanced biological activity compared to EVs obtained under normoxic conditions. The obtained EVs were collected from the conditioned medium using a sequential ultracentrifugation protocol and subsequently filtered through a 0.22 μm membrane.

[0054] In one embodiment, hypoxic MSC-EVs are characterized by a spherical morphology, a particle size of 30 to 150 nanometers, and the presence of the surface marker CD9. The vesicles encapsulate HIF-1α within their protein cargo and lack intracellular organelle markers (such as GM130), indicating high purity. These EVs are capable of modulating inflammatory signaling and epithelial fluid transport.

[0055] In one embodiment, a physiologically relevant in vitro model of virus-induced acute lung injury was used to evaluate the therapeutic effect of the EV composition. Primary human alveolar epithelial cells (AECs) were seeded onto transwell membranes and cultured, then infected with a highly pathogenic strain of influenza A (H5N1). Viral infection resulted in impaired alveolar fluid clearance (AFC), increased alveolar protein permeability (APP), and elevated expression of pro-inflammatory cytokines, including TNF-α, IFN-β, and RANTES. These pathological changes were accompanied by key ion transporters such as ENaC, CFTR, and Na+. + / K + The expression level of ATPase decreased.

[0056] In one embodiment, treatment with hypoxic MSC-EVs resulted in a significant recovery of AFC and inhibition of APP. EV-treated AECs showed increased expression of epithelial ion transporters and decreased levels of pro-inflammatory cytokines. Furthermore, this treatment induced upregulation of the antiviral gene ISG15 and reduced viral load. These effects were more pronounced with hypoxic MSC-EVs compared to normoxic EVs, indicating a stronger therapeutic effect.

[0057] In another embodiment, the therapeutic efficacy of hypoxic MSC-EV was demonstrated in vivo using a mouse model of H5N1-induced lung injury. Mice were intranasally infected with H5N1 virus and treated intravenously with either normoxic or hypoxic MSC-EV at defined time points after symptom onset. Mice treated with hypoxic MSC-EV showed better clinical outcomes, including reduced weight loss, improved survival, and lower viral titers in lung tissue. Analysis of bronchoalveolar lavage fluid (BALF) and lung homogenates revealed more significant inhibition of inflammatory cytokines, particularly TNF-α, in the hypoxic EV treatment group.

[0058] In summary, these findings demonstrate that extracellular vesicles derived from hypoxia-preregulated MSCs constitute a structurally and functionally optimized composition for the treatment of virus-induced acute lung injury. These EVs exhibit enhanced bioactivity in modulating inflammatory, epithelial, and antiviral responses, thus providing a clinically translatable cell-free therapeutic alternative for the early and late stages of respiratory viral infections.

[0059] Example

[0060] Example 1 - Materials and Methods

[0061] Virus

[0062] Influenza A virus / HK / 483 / 97 (H5N1) was used for in vitro infection. All influenza viruses were passaged in Madin-Darby canine kidney (MDCK) cells. The median tissue culture infection dose (TCID) was determined. 50 Virus titer was determined. All experiments were conducted in a biosafety level 3 facility.

[0063] Culture of UC-MSCs

[0064] Umbilical cord-MSCs were isolated by HealthBaby Biotech (Hong Kong) Company Ltd. and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) supplemented with 10% HyClone fetal bovine serum (FBS) (SV30160.03, Thermo Fisher Scientific) and 1% P / S at low glucose (1.0 g / L). UC-MSCs were cultured in a humidified atmosphere (37°C, 5% CO2), with the growth medium changed every 48–72 hours. Cells were digested with trypsin and seeded at 70% confluence for experimental use.

[0065] EV separation from UC-MSC

[0066] At 70% confluence, UC-MSCs were washed three times with HBSS to remove HyClone FBS, and then supplemented with 10% FBS-free, low-glucose DMEM. To produce hypoxic cell cultures, 100 μM cobalt(II) chloride was added to the medium. The supernatant was collected at 72 hours and stored at -20°C until EV separation. EVs were separated by differential centrifugation as follows: 1) Centrifuge at 3,000 rpm for 15 minutes at 4°C; 2) Collect the supernatant and centrifuge again at 20,000 g for 30 minutes at 4°C; 3) Collect the supernatant, pass it through a 0.22 μM filter, and centrifuge at 100,000 g for 2 hours at 4°C; 4) Discard the supernatant, wash the EV precipitate with PBS, and centrifuge the EV suspension at 100,000 g for another 2 hours at 4°C; 5) Remove the supernatant, resuspend the EV precipitate in PBS, and store at -20°C until use.

[0067] Isolation and culture of primary human AEC

[0068] AECs were isolated from non-malignant lung tissue resected from the Department of Cardiothoracic Surgery, Queen Mary Hospital, Hong Kong. First, visible bronchi were removed, and the lung tissue was minced using scissors to create fragments >0.5 mm in thickness. The minced lung fragments were washed with Hank's balanced salt solution (Invitrogen, USA) at pH 7.4 to partially remove macrophages and blood cells. Then, the washed lung fragments were digested for 40 minutes at 37°C in a shaking water bath with 0.5% trypsin (GibcoBRL, USA) and 4 U / ml elastase (Worthington Biochemical, USA). Digestion was terminated with 40% FBSDMEM / F12 medium and DNase I (350 units / ml) (Sigma, USA). Incompletely digested lung cells were isolated using a 50 μM pore size cell filter (BD Bioscience, USA). Cell clumps were dispersed by repeated pipetting for 10 minutes. After decelerating rotation, cells were incubated with a 1:1 mixture of DMEM / F12 medium containing 5% FBS and 350 units / ml DNase I and small airway growth medium (SAGM) (Lonza, USA). Cells were seeded onto new tissue culture flasks (Corning, USA) for adhesion at 37°C. Unadhered cells were collected for centrifugation and resuspended in new tissue culture flasks with SAGM. The medium was changed daily for the first 4 days of plating. AECs were trypsinized at 75% confluence for seeding.

[0069] In vitro acute lung injury model

[0070] To investigate the effects of influenza virus on alveolar fluid clearance and protein permeability in human alveolar epithelial cells, a physiologically relevant 24-transwell in vitro acute lung injury model was used. Alveolar epithelial cells were plated onto the top side chambers of a 24-well Costa Transwell insert (0.4 μm pore size, Corning), at a density of 1 × 10⁻⁶ cells per well. 5 Cells. A microporous transwell membrane established a liquid-liquid interface similar to that of human lung epithelium, and the plated cells were maintained in a humidified atmosphere (5% CO2, 37°C). Transepithelial resistance was maintained at ≥800 Ω / cm.2 This indicates that there is good tight junction integrity between cells.

[0071] Measurement of alveolar fluid clearance and alveolar protein permeability

[0072] Net alveolar fluid transport from the top side chamber of the transwell culture insert (containing a monolayer of influenza virus-infected alveolar epithelial cells) to the basolateral chamber of the transwell was measured 24 hours post-infection. Alveolar epithelial cells were inoculated with the corresponding influenza A virus at a multiplicity of infection (MOI) of 0.1 for 1 hour. Then, 200 μl of FITC-labeled dextran (Sigma-Aldrich) at a size of 70 kDa was added to the cells (final concentration 500 ng / μl). After 5 minutes, 100 μl of the sample was collected from the top side chamber for initial FITC measurement and then transferred back to the top side chamber for overnight incubation. After 24 hours of dextran incubation, 100 μl was collected from both the top side chamber and the basolateral chamber for final FITC measurement. Fluorescence of each sample was measured using a modulus fluorometer (FLUOstar OPTIMA, BMG Labtech) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. A standard curve with known FITC concentrations was constructed to calculate the presence of FITC-glucan in the transwell chamber at different time points. Net alveolar fluid transport across the epithelial monolayer was calculated as [1 - (FITC concentration in the initial apical sample / FITC concentration in the final apical sample)] / 200 μl / 0.33 cm⁻¹. 2 / 24 hours. Final basolateral readings were collected after 24 hours of dextran incubation to measure protein permeability based on the unidirectional flux of fluorescently labeled dextran from the top side of the transwell culture insert to the basolateral chamber.

[0073] Viral infection in mice

[0074] Female BALB / c mice aged 6-8 weeks were intranasally inoculated with 25 μl of 10-dose A / Hong Kong / 486 / 1997 (H5N1) influenza virus. 6 log TCID 50 Mice were intravenously injected with 100 μl of 1×10⁻⁶ solution on days 3, 5, and 7 post-infection. 8MSC-EV or PBS were administered. Survival and body weight were monitored for 10 days. On day 10 post-infection, the virus was titrated in the lungs of three mice in each group. BAL solution was collected using a mouse Luminex assay for cytokine assays, following the manufacturer's instructions (R&D Systems, USA). Additionally, lung tissue from three mice in each group was fixed for histopathological analysis.

[0075] Quantitative RT-PCR analysis was performed on influenza matrix genes, inflammatory cytokines, antiviral genes, and ion transporters. mRNA

[0076] Total RNA and cellular proteins were extracted from infected AECs 24 hours post-infection. RNA extraction was performed according to the manufacturer's instructions, and then reverse transcription of RNA to complementary DNA was performed using the PrimeScript RT kit (TaKaRa, Dalian). AECs and MSCs infected with influenza virus at MOI 2 were lysed with 350 μl of buffer RLT (Qiagen, Germany) containing β-mercaptoethanol (Sigma-Aldrich, USA). RNA extraction was performed using the MiniBEST Universal RNA Extraction Kit (TaKaRa, Dalian), with DNase treatment performed according to the manufacturer's instructions. The PrimeScript RT kit (TaKaRa, China) was used for reverse transcription. Via TM A real-time PCR system (Applied Biosystems, USA) was used for real-time PCR. Gene expression profiles were normalized using the housekeeping gene β-actin mRNA. Standard plasmids with known copy numbers were run with the genes under study to generate a standard curve to determine absolute copy numbers. The master mix (including Power SYBR Green PCR master mix (Applied Biosystems, USA), in which cDNA was reverse transcribed from 500 ng of total RNA) was amplified using an ABI 7500 PCR system (Applied Biosystems, USA) for 40 cycles via real-time PCR. Gene expression and statistical analysis were performed according to the manufacturer's instructions.

[0077] Statistical analysis

[0078] All in vitro experiments were performed independently in triplicate. Groups were compared using an unpaired two-tailed t-test. A difference was considered significant if p ≤ 0.05.

[0079] Example 2 - Isolation of MSC-EVs from MSC secretome

[0080] Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) were cultured under normoxic (21% O2) or chemically induced hypoxia conditions to investigate the effect of oxygen availability on extracellular vesicle (EV) production. Hypoxia was induced by supplementing the medium with 100 μM cobalt(II) chloride (CoCl2) (a well-established hypoxia mimic). The medium was prepared using low-glucose Duchenne modified Ehrlich medium (DMEM) supplemented with 10% EV-depleted fetal bovine serum (FBS). Cells were incubated under the appropriate conditions for 72 hours prior to EV collection. Figure 1A A schematic representation of the EV separation procedure is shown.

[0081] After incubation in hypoxic or normoxic conditions, the conditioned medium containing secreted EVs and soluble proteins is collected, and the conditioned medium is subjected to a multi-step differential centrifugation process to selectively separate the EVs, such as... Figure 1A Demonstration. This purification strategy aims to maximize the yield and purity of small-sized EVs while minimizing contamination from cell debris and soluble protein aggregates. The separation procedure includes: (i) Centrifuge at 3,000 rpm for 15 minutes at 4°C to remove cell debris and apoptotic bodies; (ii) Centrifuge at 20,000×g for 30 minutes at a medium speed to eliminate larger vesicles and macromolecular complexes; (iii) Filtering the supernatant through a membrane with a pore size of 0.22 μm to remove particles larger than the EV size range; and (iv) Centrifuge at 100,000×g for 2 hours to precipitate EV.

[0082] To ensure the removal of residual soluble proteins and further concentration of EVs, the precipitate was washed once with phosphate-buffered saline (PBS) and subjected to a second round of ultracentrifugation under the same conditions. The final EV precipitate was resuspended in PBS and stored at -20°C until used for downstream characterization and functional studies. This method combines density-based and particle size-based sequential fractionation with a two-step ultracentrifugation process, enabling highly reproducible EVs with intact morphology and minimal contamination. Compared to conventional single-step ultracentrifugation methods, the method described in this invention significantly improves EV purity and experimental consistency, which is of great significance for subsequent molecular characterization analysis and therapeutic applications.

[0083] Characterization of the separated EVs confirmed the successful acquisition of small EVs with high purity and functional activity. For example... Figure 1BAs shown, Western blot analysis revealed that the canonical EV marker CD9 was strongly expressed in both normoxic and hypoxic EVs, while the endoplasmic reticulum protein GM130 (a known negative marker of EV contamination) was absent in all EV formulations. The absence of GM130 confirmed the effective exclusion of intracellular organelles and validated the purity of the obtained EV components. Simultaneously, HIF-1α expression was observed in cell lysates and corresponding hypoxic EVs, confirming the effective induction of hypoxia preconditioning in MSCs and suggesting that functional hypoxia-related proteins may be carried into secreted EVs. β-actin was used as an internal loading control.

[0084] The particle size distribution and concentration of the separated EVs were determined using nanoparticle tracking analysis (NTA). Figure 1C The particle population exhibited a unimodal size distribution centered at approximately 120.6 nm, with a full width at half maximum (FWHM) of 113.9 nm. The particle concentration was determined to be 6.6 × 10⁻⁶. 6 The particle / mL count indicates robust EV yield. Notably, the size range is consistent with the definition of small EVs (typically <150 nm), and the sharp peaks indicate a highly homogeneous vesicle population, which enhances batch-to-batch reproducibility for therapeutic applications.

[0085] Transmission electron microscopy (TEM) further confirmed the vesicle morphology and ultrastructural integrity of the isolated particles. Figure 1D The EVs exhibit a spherical, lipid bilayer-closed structure with a diameter of less than 150 nm, consistent with exosome-like morphology. High-resolution imaging at 100 kV and ×28,500 magnification also revealed the absence of vesicle aggregation or membrane rupture, further supporting the structural quality and physical stability of the EV formulation.

[0086] In summary, the combined biochemical (Western blot), biophysical (NTA), and TEM analyses validated that the EVs isolated using the published protocol exhibited key physicochemical characteristics consistent with those of functional small EVs. Furthermore, the presence of hypoxia-inducible proteins within hypoxia-originating EVs provides molecular evidence for their enhanced bioactivity, supporting their potential use as a novel class of therapeutically active nanovesicles with superior potency compared to unregulated EVs.

[0087] Example 3 - Hypoxic MSC-EV in vitro recovery of AFC and APP

[0088] To evaluate the therapeutic effect of MSC-EV, a physiologically relevant in vitro model of influenza virus-induced alveolar injury was established, such as... Figure 2AAs shown in the diagram. In this model, primary human alveolar epithelial cells were seeded on the top side of a porous transwell culture insert and cultured to form a monolayer structure mimicking the human alveolar barrier. The cells were grown to a confluence state under gas-liquid interface conditions, and the transepithelial electrical resistance (TEER) measured a value exceeding 800 Ω·cm. 2 This verifies that it possesses a complete and tightly connected structure. This threshold indicates the formation of a functionally intact epithelial barrier, suitable for simulating directional fluid transport and epithelial leakage processes.

[0089] The experimental setup allows for controlled viral damage and therapeutic intervention on the top side of an AEC monolayer, thus mimicking the physiological orientation of exposure to airborne respiratory pathogens and therapeutic delivery. Specifically, H5N1 virus and MSC-EVs were directly administered into the top-side chamber, enabling simultaneous infection and treatment in a space-constrained and reproducible environment. Compared to conventional immersion culture methods, this model offers several advantages, including the ability to perform targeted cytokine sampling, quantitative analysis of alveolar fluid clearance (AFC) and alveolar protein permeability (APP), all clinically relevant key endpoints in acute lung injury. The use of primary human AECs in the transwell insert system significantly improves biological fidelity compared to traditional in vitro methods relying on undifferentiated or immortalized cell lines, making it a robust platform for assessing the barrier restoration, immunomodulatory, and antiviral properties of therapeutic EVs in the context of viral lung epithelial damage.

[0090] To assess the impact of MSC-derived EVs on epithelial barrier function following viral damage, primary human alveolar epithelial cells were infected with highly pathogenic influenza A / H5N1 virus at a multiplicity of infection (MOI) of 0.1. Key physiological parameters, including alveolar foci (AFC) and active pharmaceutical ingredient (APP), reflecting the integrity and function of the alveolar barrier, were measured 24 hours post-infection.

[0091] like Figure 2B As shown, H5N1 infection significantly reduced AFC, indicating impaired ion transport and fluid reabsorption across the epithelial monolayer. Conversely, treatment with MSC-EV restored AFC to baseline levels. Notably, hypoxia-preconditioned MSCs (EV-Hyp) demonstrated a significantly stronger effect on AFC recovery compared to normoxic EVs (EV-Nor), with AFC values ​​even exceeding those of the uninfected control group. This unexpected enhancement suggests that hypoxic EVs not only alleviate viral damage but also promote active fluid transport more effectively than standard EV formulations. Similarly, as measured by the percentage change in FITC-glucan translocation (…),… Figure 2CThe study found that H5N1 infection significantly increases alveolar protein permeability. This reflects disruption of epithelial tight junctions and mimics the pathophysiology of acute lung injury. Treatment with MSC-EVs significantly reduced protein leakage, with hypoxic-origin EVs showing the most significant inhibition of APP (anti-inflammatory pulmonary artery closure) across all tested groups. This reduction was statistically significant compared to the untreated group and the EV-Nor group, further highlighting the functional advantage of hypoxic EVs in restoring epithelial barrier integrity. These findings demonstrate that hypoxic-preconditioned MSC-EVs provide measurable and unexpectedly enhanced therapeutic effects on two clinically relevant epithelial functions, fluid absorption and barrier tightness, which are often impaired during severe respiratory viral infections. The dual restoration of AFC (air filtration capacity) and APP highlights the multiple mechanisms of action of hypoxic MSC-EVs and distinguishes them from conventional EV treatments in terms of efficacy and physiological relevance.

[0092] H5N1 infection also leads to the upregulation of pro-inflammatory cytokines (including TNF-α, IFN-β, and RANTES) and ion transporters (such as ENaC, CFTR, and Na+). + / K + Downregulation of ATPases, etc. MSC-EV treatment reversed these gene expression changes, with hypoxic MSC-EV inducing more pronounced suppression of cytokines and restoration of transporter expression. Figure 2D-2E Furthermore, MSC-EV upregulated the expression of antiviral genes, including ISG15, with hypoxic EV again showing a stronger induction effect than normoxic EV. Figure 2F Both EV groups suppressed viral titers in infected AECs, but hypoxic MSC-EVs exhibited significantly stronger antiviral activity. Figure 2G ).

[0093] These findings confirm that hypoxic MSC-EVs possess enhanced therapeutic activity in vitro, restoring epithelial function, reducing inflammation, and activating antiviral responses in influenza-infected human alveolar epithelial cells. The magnitude and breadth of these effects are unexpected, as conventional MSC or EV interventions in existing technologies have not revealed or implied such effects.

[0094] Example 4 - In vivo therapeutic evaluation of hypoxic MSC-EVs in mice infected with H5N1

[0095] To further demonstrate the therapeutic potential of hypoxia-preregulated MSC-derived extracellular vesicles (hypoxic MSC-EVs) in physiologically relevant disease contexts, an in vivo mouse model of virus-induced acute lung injury was established, such as... Figure 3A As shown. Female BALB / c mice aged 6–8 weeks were intranasally inoculated under mild anesthesia with 25 μL of 10... 6 TCID 50The highly pathogenic human-derived influenza A virus strain A / Hong Kong / 486 / 1997 (H5N1) is used. This route of infection accurately mimics the natural transmission of airborne respiratory viruses and selectively targets the lower respiratory tract, including the alveolar regions.

[0096] Following infection, animals were monitored for clinical symptoms such as weight loss, respiratory distress, and decreased activity. The appearance of obvious symptoms in mice represented a clinically relevant treatment window, rather than a preventative environment. Mice were administered MSC-EVs intravenously on days 3, 5, and 7 post-infection. Treatment groups included EVs derived from normoxic (EV-Nor) or hypoxic-preconditioned (EV-Hyp) MSC cultures, with a uniform dosage of 1 × 10⁻⁶ EVs per injection. 8 Individual particles. This post-symptom administration strategy, which differs from previous preventative EV administration methods, better reflects a more rigorous and translational assessment of therapeutic efficacy. Furthermore, the systemic (intravenous) delivery route allows EVs to circulate and reach the pulmonary vascular system, thereby promoting targeted uptake in inflamed or damaged alveolar tissue. In a tightly controlled mouse model, the combination of highly virulent viral strains, concurrent clinical treatment initiation time, and comparative EV subtypes not only assessed survival benefits but also the mechanistic effects of hypoxic MSC-EVs on inflammation resolution, viral clearance, and lung tissue recovery.

[0097] Body weight and survival were monitored for 10 days post-infection. Mice treated with hypoxic MSC-EV showed significantly reduced body weight loss compared to untreated controls. Figure 3B ), and showed a higher survival rate during the observation period ( Figure 3C Inflammatory cytokine levels were measured in lung homogenates and bronchoalveolar lavage (BAL) fluids. Results showed that MSC-EV treatment reduced H5N1-induced pro-inflammatory responses, with hypoxic MSC-EV exhibiting a more significant inhibitory effect, particularly in TNF-α expression. Figure 3D-3E ).

[0098] Viral load was assessed using M-gene expression analysis and tissue culture infectious dose (TCID) assay. Both normoxic MSC-EV and hypoxic MSC-EV effectively inhibited viral replication in lung tissue, with hypoxic MSC-EV showing superior antiviral efficacy. Figure 3F-3G ).

[0099] These in vivo results confirm that hypoxia-preconditioned MSC-EVs not only alleviated clinical symptoms and reduced mortality in mice infected with H5N1, but also more effectively reduced lung inflammation and viral load than normoxic MSC-EVs. These results strongly demonstrate that hypoxia-preconditioned MSC-EVs have significantly enhanced therapeutic efficacy in treating in vivo respiratory virus-induced acute lung injury.

[0100] Example 5 - Proteomics characterization of hypoxia-preregulated MSC-EVs

[0101] To elucidate the molecular basis of the enhanced therapeutic efficacy of hypoxia-preregulated MSC-derived extracellular vesicles (hypoxic MSCs-EVs), comparative proteomics analysis was performed using mass spectrometry. EV samples from both normoxic and hypoxic MSC cultures were analyzed to identify differentially expressed proteins and their associated biological functions and localizations. Figures 4A-4C ).

[0102] Proteomics analysis revealed significant differences in protein cargo between hypoxic MSC-EVs and normoxic MSC-EVs. Several proteins were significantly upregulated in the hypoxic EV group, many of which are associated with immune regulation, antiviral responses, cellular stress resistance, and tissue regeneration. Figure 4A These proteins were not observed at comparable levels in normoxic EVs, indicating that hypoxia preconditioning selectively enriches bioactive components associated with lung injury repair.

[0103] Gene ontology (GO) analysis further revealed that the upregulated proteins in hypoxic MSC-EVs were mainly located in extracellular vesicles, the cytoplasmic membrane, and exosome compartments. Figure 4B From a functional perspective, these proteins are involved in key biological processes, including cytokine binding, receptor-mediated signaling, wound healing, regulation of oxidative stress, and RNA binding. Figure 4C ).

[0104] The proteomic differences observed between hypoxic and normoxic MSC-EVs provide molecular-level validation of the enhanced functional efficacy of hypoxic EVs in in vitro and in vivo experiments. Notably, based on previous studies of normoxic MSC-EVs, the differential expression profiles are unpredictable, highlighting the importance of hypoxia regulation in modulating the therapeutic potential of EVs. These findings support the innovative concept of this invention, namely, that by simply pretreating MSCs under hypoxic conditions, EVs with both structural and functional differentiation and enhanced regenerative and antiviral potential can be obtained.

[0105] The invention has been provided above for illustrative and descriptive purposes. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations will be apparent to those skilled in the art.

[0106] The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention with respect to the various embodiments and with various modifications suitable for the particular purpose considered.

Claims

1. An extracellular vesicle (EV) composition, characterized in that, The EV composition comprises an EV population isolated from one or more mesenchymal stromal cells (MSCs) cultured under chemically induced hypoxia conditions, wherein the one or more MSCs are exposed to a hypoxia mimicry prior to collection of the EV population, the isolated EV population exhibits a particle size distribution in the range of 30 nm to 150 nm in diameter, and the isolated EV population is characterized by a lipid bilayer membrane encapsulating cytoplasmic protein cargo, wherein the cytoplasmic protein cargo contains at least CD9 and hypoxia-inducible factor 1-α (HIF-1α) and is free of GM130.

2. The EV composition according to claim 1, wherein the MSC is umbilical cord-derived mesenchymal stromal cells.

3. The EV composition according to claim 1, wherein the one or more MSCs are exposed to 50 μM to 200 μM cobalt(II) chloride for 48 to 96 hours, or alternatively to hypoxia mimics such as 50 μM to 100 μM deferoxamine or 0.5 mM to 1 mM dimethyl oxaloyl glycine.

4. The EV composition according to claim 1, wherein the EV population is obtained from conditioned medium by sequential ultracentrifugation and 0.22 μm membrane filtration.

5. The EV composition of claim 1, wherein the isolated EV composition comprises at least one protein selected from the group consisting of: apolipoprotein E (APOE), fibrinogen γ chain (FGG), and neuronal pentameric protein-1 (NPTX1), wherein the expression level of the at least one protein is at least 2-fold higher than that of EVs derived from normoxic cultured MSCs.

6. The EV composition of claim 1, wherein when the EV composition acts on alveolar epithelial cells infected with highly pathogenic influenza A (H5N1), it reduces the mRNA expression level of at least one cytokine selected from TNF-α, IFN-β and RANTES by at least 2-fold relative to untreated infected cells.

7. The EV composition according to claim 1, wherein the expression of ion transporters selected from the group consisting of: epithelial sodium channels (ENaC), CFTR, and Na+ is regulated on the EV composition. + / K + ATPase.

8. The EV composition of claim 1, wherein the EV composition induces at least 2-fold ISG15 mRNA expression in virus-infected alveolar epithelial cells.

9. The EV composition of claim 1, wherein the EV is further formulated in a pharmaceutically acceptable carrier suitable for intravenous administration.

10. The EV composition of claim 1, wherein the EV is formulated into a dosage form selected from the group consisting of: The freeze-dried powder contains one or more cryoprotectants selected from the group consisting of trehalose, sucrose, and mannitol; Atomizable aqueous solution, wherein the atomizable aqueous solution is formulated for lung administration; and One or more polymeric microparticles, the one or more polymeric microparticles comprising the EV encapsulated in a biodegradable polymer selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), chitosan, and hyaluronic acid.

11. A method for treating acute lung injury in a subject of need, characterized in that, The method comprises administering to the subject an effective amount of the EV composition according to claim 1, wherein the lung injury is caused by a viral infection, and wherein the EV is derived from mesenchymal stromal cells (MSCs) cultured under chemically induced hypoxia using a hypoxia mimic.

12. The method of claim 11, wherein the MSC is exposed to 50 μM to 200 μM cobalt(II) chloride for 48 to 96 hours.

13. The method of claim 11, wherein the EV composition is administered via intravenous, intranasal, or intratracheal route.

14. The method of claim 11, wherein the EV can reduce the viral load in lung tissue by at least 50% compared to an untreated control group.

15. The method of claim 11, wherein the EV reduces the expression levels of TNF-α and RANTES by at least 2-fold compared to the untreated control group.

16. The method of claim 11, wherein the EV restores alveolar fluid clearance (AFC) and reduces alveolar protein permeability (APP) within 24 hours after administration.

17. The method of claim 11, wherein the EV increases ISG15 expression in infected alveolar epithelial cells by at least 2-fold.

18. The method of claim 11, wherein the EV composition is administered in combination with one or more antiviral agents, the one or more antiviral agents comprising oseltamivir, zanamivir, or ribavirin.

19. The method of claim 11, wherein the effective amount of the EV composition comprises approximately 1 × 10⁻⁶ per application. 8 Each EV particle up to 1×10 12 Dosage of each EV particle.

20. The method of claim 11, wherein the EV composition is administered in multiple doses over a period of 3 to 10 days after the onset of viral infection symptoms.

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